CN111821514B - Silk sericin composite membrane and preparation method thereof - Google Patents
Silk sericin composite membrane and preparation method thereof Download PDFInfo
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Abstract
The invention provides a preparation method of a silk fibroin and sericin composite membrane, which comprises the following steps: A) placing silk in warm water for pretreatment, then placing the silk in 65-95 ℃ water for treatment, and drying to obtain primary treated silk; B) treating the primarily treated silk in a neutral salt solution, and purifying the obtained silk fibroin sericin composite dissolved solution to obtain a silk fibroin sericin composite solution; C) and concentrating the silk fibroin and sericin composite solution, forming a film, placing the obtained initial composite film in micromolecular monohydric alcohol for treatment, and then cleaning and drying to obtain the silk fibroin and sericin composite film. The silk fibroin sericin composite solution is prepared by adopting a mild degumming and one-step dissolving method, the composite membrane prepared by the silk fibroin sericin composite solution has high strength and high toughness, and the technical problem of neck clamping which is insufficient in mechanical property and is derived from natural biopolymers for repairing hard tissues such as dura mater and dura mater is solved.
Description
Technical Field
The invention relates to the technical field of biomedical materials, in particular to a silk fibroin sericin composite membrane and a preparation method thereof.
Background
The structure of natural dura mater and dura mater is a firmer connective tissue film, and the currently applied dura mater and dura mater repairing materials are from autologous tissues, allogenic bodies, xenogenic biological materials, artificial synthetic materials and the like, mainly import products and are expensive. The best treatment effect of autologous tissue and allogeneic transplantation exists, but the source is limited, and the serious restriction exists on a plurality of factors such as supply area damage and complications. In clinical practice, the foreign biological materials such as bovine pericardium, sheep pericardium, porcine peritoneum, mesentery and the like are used for replacing the foreign biological materials, the sources are wide, the price is low, but certain toxicity is remained due to the treatment of foreign antigen protein, and the cell growth is prevented.
The concept of tissue engineering was proposed in the 90 s of the 20 th century, and then the research of scaffold materials for tissue engineering was rapidly developed. The artificial dura mater and dura mater should have smooth surfaces, excellent strength, toughness and compactness, and excellent biocompatibility. The synthetic polymer material has the advantages of wide source, controllable physicochemical properties, excellent processing formability and the like, such as various synthetic polymers of expanded polytetrafluoroethylene, polycaprolactone and the like, but the material lacks bioactivity and has poor cell compatibility, and the microenvironment of tissues is not influenced by degradation or degradation products. Since natural polymers have excellent biological activity, they are widely favored in the research of tissue engineering materials, and collagen, chitosan, fibroin, and the like have been used in the construction research of hard tissues. However, most of the materials have poor mechanical properties, and the application of the materials as hard tissue implants has great challenges.
In contrast to other natural polymers, especially protein polymers: the silk is composed of fibroin fibers (the weight ratio is 70-80%) and sericin outside the silk fibers (the weight ratio is 20-30%), the composition is unique, the protein purity is very high, the preparation method can be simple and mild, small damage to fibroin macromolecules can be controlled, and the silk fibroin composite material is not like collagen and other natural macromolecules which are complex in purification, small in molecular weight, scattered in distribution, easy to change and the like. The silk fibroin macromolecule is connected by 6 heavy chains with 390kDa and 6 light chains with 26kDa in disulfide bonds, and is combined with another glycoprotein/P25 with 25kDa in a hydrophobic interaction manner, wherein the heavy chains have a sequence structure basis forming a compact-structure and high-performance material. The molecular weight of sericin is also larger, more than 9 sericins exist, relating to Chuanminglan points out the outer, middle and inner three layers of sericin from the histological angle, and the small loose meter suggests that the outer sericin has 4 types from the outside and the inner, namely sericin I, sericin II, sericin III and sericin IV, and the solubility property from the outside and the inner is lower and lower, and the beta conformation is increased.
Silk fibroin and sericin have excellent cell compatibility, blood compatibility and tissue compatibility and controllable molding, and have been widely studied in tissue engineering applications such as skin, blood vessels, bones and the like as a scaffold material for tissue engineering. The regenerated silk fibroin solution is obtained by dissolving strong alkali weak acid salt or enzyme degumming salt, and the methods cause the damage of silk fibroin macromolecules; the regenerated sericin solution is mainly from silkworm cocoons, and the two solutions are generally prepared by independent separation. For example, CN02138129.1 discloses a preparation method of a silk fibroin and sericin composite tissue engineering scaffold, wherein a composite material is prepared by mixing pre-prepared regenerated silk fibroin and regenerated sericin in proportion, a degumming method of silk fibroin adopts a strong base and weak acid salt solvent, and sericin extracts a part of an outer layer of silkworm cocoon silk which is externally coated with sericin. For example, CN201510353363.8 discloses a scaffold material for bone tissue engineering comprising silk fibroin and sericin and a preparation method thereof, wherein regenerated silk fibroin and regenerated sericin are respectively obtained step by step and then mixed to prepare the scaffold material, wherein the preparation of silk fibroin does not disclose specific process conditions, and the sericin is obtained from silkworm cocoon and treated with organic solvents such as alcohol. In addition, for example, CN201010288275.1 and CN201410651322.2 disclose the preparation of silk fibroin films, wherein degumming of silk fibroin fibers also employs a strong base and weak acid salt degumming method.
The regenerated silk (fibroin, sericin) protein film researched by the above is also poor in mechanical property, and is difficult to meet the requirement of replacing hard tissues such as dura mater, dura mater and the like, the key of the problem is that the existing degumming method and extraction method have large damage to the macromolecular structures of fibroin and sericin, and the polymerization degree (molecular weight) of macromolecules is an extremely important intrinsic factor for determining the performance of macromolecules.
Disclosure of Invention
The invention aims to provide a preparation method of a silk fibroin sericin composite membrane, and the silk fibroin sericin composite membrane has high strength and high toughness.
In view of this, a method for preparing a silk fibroin and sericin composite membrane comprises the following steps:
A) placing silk in warm water for pretreatment, then placing the silk in 65-95 ℃ water for treatment, and drying to obtain primary treated silk;
B) treating the primarily treated silk in a neutral salt solution, and purifying the obtained silk fibroin sericin composite dissolved solution to obtain a silk fibroin sericin composite solution;
C) and concentrating the silk fibroin and sericin composite solution, forming a film, placing the obtained initial composite film in micromolecular monohydric alcohol for micro-treatment, and then cleaning and drying to obtain the silk fibroin and sericin composite film.
Preferably, the temperature of the warm water is 35-60 ℃, and the pretreatment time is 1-10 min.
Preferably, in the step A), the treatment time in the water at 65-95 ℃ is 1-8 h.
Preferably, in the step a), the ratio of the silk to the warm water is 1 g: (40-60) ml, wherein the ratio of the pretreated silk to water is 1 g: (40-60) ml.
Preferably, step a) further comprises, after the pretreatment: taking out the pretreated silk, washing in water at 25-30 ℃, and dehydrating;
the method also comprises the following steps of: washing the treated silk in water at 25-30 ℃ and then dehydrating; the drying is carried out by hot air at 40-60 ℃.
Preferably, the neutral salt solution is 8-10M of lithium bromide aqueous solution, and the ratio of the primarily treated silk to the lithium bromide aqueous solution is 1 g: (20-30) ml.
Preferably, in the step B), the treatment temperature is 50-80 ℃.
Preferably, the purification method specifically comprises:
and (3) filling the obtained silk fibroin sericin composite dissolved solution into a dialysis bag with the molecular weight cutoff of 10-50 kDa, and placing the dialysis bag in deionized water for continuous dialysis for 1-3 days.
Preferably, in the step C), the small molecular monohydric alcohol is an ethanol solution with the concentration of 70-80%, and the micro-processing time is 10-60 min.
The application provides a silk fibroin sericin composite membrane, which is prepared from a silk fibroin sericin composite solution, and the preparation method of the silk fibroin sericin composite solution comprises the following steps:
A) placing silk in warm water for pretreatment, then placing the silk in 65-95 ℃ water for treatment, and drying to obtain primary treated silk;
B) and (3) treating the primarily treated silk in a neutral salt solution, and purifying the obtained silk fibroin and sericin composite dissolved solution to obtain the silk fibroin and sericin composite solution.
The application provides a preparation method of a silk fibroin composite membrane, which adopts mild degumming and a one-step dissolving method to prepare a silk fibroin composite solution, and adopts pretreatment in warm water and subsequent treatment in water at a specific temperature to remove part or all of sericin on the surface of silk, wherein the existence of sericin increases strong hydrogen bond combination and network structure among molecular chains of the composite membrane, thereby improving the strength and toughness of the composite membrane, or adopts degumming in water at a specific temperature, and the silk fibroin macromolecular structure is protected in the subsequent degumming process of silk fibroin, so that the regenerated silk fibroin has high molecular weight to improve the strength of the composite membrane; the sericin of the silk fibroin fiber outer layer is different from the outer layer and the inner layer along with the coating layer, the sericin type, structure and performance are different, the high-strength and high-toughness silk fibroin composite membrane with different compositions and controllable mechanical property is obtained by controlling the degumming degree, the technical problem of a clamping neck with insufficient mechanical property of a tissue repair material from natural biopolymers for repairing hard tissues such as dura mater, dura mater spinalis and the like is solved, the composite membrane has the activity of inducing cells to be rapidly adhered and grown in situ and promoting tissue regeneration, and meanwhile, the composite membrane can be biodegraded and absorbed.
Detailed Description
For a further understanding of the invention, reference will now be made to the preferred embodiments of the invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the invention, and not to limit the scope of the claims.
Aiming at the problem that silk fibroin macromolecules are seriously damaged or silk fibroin and sericin composite solutions are respectively prepared to influence the toughness and the strength of the silk fibroin and sericin composite membrane in the prior art, the application provides the silk fibroin and sericin composite membrane, the composite membrane is prepared from the silk fibroin and sericin composite solutions, in the preparation process of the silk fibroin and sericin composite solutions, the silk fibroin and sericin composite solutions with high molecular weight and high viscosity are prepared by adopting a deionized water mild degumming and one-step dissolving method, the method can avoid the damage of the silk fibroin and sericin macromolecules, and finally the silk fibroin in the prepared silk fibroin and sericin composite solution has high viscosity and high molecular weight, so that the silk fibroin and sericin composite membrane prepared by the method has good toughness and strength. Specifically, the embodiment of the invention discloses a preparation method of a silk fibroin sericin composite membrane, which comprises the following steps:
A) placing silk in warm water for pretreatment, then placing the silk in 65-95 ℃ water for treatment, and drying to obtain primary treated silk;
B) treating the primarily treated silk in a neutral salt solution, and purifying the obtained silk fibroin sericin composite dissolved solution to obtain a silk fibroin sericin composite solution;
C) and concentrating the silk fibroin and sericin composite solution, forming a film, placing the obtained initial composite film in micromolecular monohydric alcohol for micro-treatment, and then cleaning and drying to obtain the silk fibroin and sericin composite film.
In the preparation process of the silk fibroin and sericin composite membrane, firstly, placing silk in warm water for pretreatment, then placing the silk in water at 65-95 ℃ for treatment, and drying to obtain the primarily treated silk; in the process, sericin of the silk is completely or partially dissolved, the fibroin is still in a fiber state, if undissolved sericin exists, the undissolved sericin is wrapped on the outer surface of the fibroin fiber, and the dissolved sericin exists in water and is in a solution state. The temperature of the warm water is 35-60 ℃, namely the silk can be pretreated in the warm water at 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃ or 60 ℃, and the ratio of the silk to the warm water is 1 g: (40-60) ml, more specifically, the ratio of the silk to the warm water is 1 g: 42ml, 1 g: 45ml, 1 g: 48ml, 1 g: 52ml, 1 g: 55ml, 1 g: 58 ml. The pretreatment is carried out in the constant-temperature warm water for 1-10 min, and the pretreatment is carried out in stirring all the time so as to ensure that the silk can be preliminarily and completely immersed in the water. And after the pretreatment is finished, taking out the pretreated silk, washing the silk in deionized water at 25-30 ℃ and dehydrating the silk. The dehydrated silk is treated in water at 65-95 deg.C, more specifically, the dehydrated silk is treated in water at 68 deg.C, 70 deg.C, 72 deg.C, 75 deg.C, 78 deg.C, 80 deg.C, 82 deg.C, 84 deg.C, 87 deg.C, 90 deg.C or 93 deg.C. This application silk is at the mode of aquatic preliminary treatment again in water treatment earlier, can dissolve sericin in the undamaged while of protection fibroin macromolecular structure. The ratio of the pretreated silk to water is 1 g: (40-60) ml, more specifically, the ratio of the pretreated silk to water is 1 g: 42ml, 1 g: 45ml, 1 g: 48ml, 1 g: 50ml, 1 g: 52ml, 1 g: 55ml, 1 g: 58ml or 1 g: 60 ml. The degumming rate of the silk sericin can be adjusted according to different temperatures and time of water so as to realize the preparation of silk sericin composite solutions with different viscosities and different molecular weights; in the application, the time for treating the silk in the water is preferably 1-8 hours, so that the silk is partially or completely degummed; more specifically, the time for the treatment in water is 1.5h, 2h, 2.5h, 3h, 3.2h, 3.6h, 4.2h, 4.5h, 5.0h, 5.5h, 6.0h, 6.5h, 6.8h, 7.0h or 7.5 h. After treatment, the treated silk is washed in water at 25-30 ℃, dehydrated and dried; the drying is carried out by hot air at 40-60 ℃. The silk is subjected to the treatment to obtain the primary treated silk, the silk still presents a fiber state, the protection of the silk fibroin macromolecules is realized, and the viscosity of the silk fibroin sericin is ensured.
According to the invention, the silk after primary treatment is treated in a neutral salt solution and purified, and then the silk fibroin sericin composite solution is obtained. In the process, the primary-treated fibrous silk is dissolved to obtain the silk fibroin sericin composite solution, wherein the inner core of the silk is silk fibroin fibers, and the periphery of the silk fibroin fibers is wrapped with sericin with different contents. Neutral salt solution specifically is selected from the lithium bromide aqueous solution in this application, and its concentration is 8 ~ 10M, the silk of preliminary treatment with the proportion of lithium bromide aqueous solution is 1 g: (20-30) ml, more specifically, the proportion of the primary treated silk rain to the lithium bromide aqueous solution is 1 g: 22ml, 1 g: 24ml, 1 g: 25ml, 1 g: 27ml or 1 g: 29 ml. The treatment temperature is specifically 50-80 ℃, and more specifically, the treatment temperature is specifically 52 ℃, 57 ℃, 62 ℃, 65 ℃, 67 ℃, 68 ℃, 72 ℃, 75 ℃ or 78 ℃.
After the treatment, purifying the obtained silk fibroin sericin dissolving solution to remove lithium bromide and small molecular weight silk fibroin; the purification method specifically comprises the steps of filling the obtained silk fibroin and sericin composite dissolved solution into a dialysis bag with the molecular weight cutoff of 10-50 kDa, and placing the dialysis bag in deionized water for continuous dialysis for 1-3 days.
The method comprises the steps of preparing a silk fibroin sericin composite solution, concentrating the silk fibroin sericin composite solution to a certain degree, removing bubbles, pouring the solution into a flat plate, performing rotary air drying at room temperature, performing micro-treatment on the obtained initial composite membrane in a micromolecular monohydric alcohol solution for 10-60 min to ensure that the obtained composite membrane is insoluble in water and tissue fluid, finally soaking the composite membrane in water, and performing constant-temperature and constant-humidity air drying to obtain the silk fibroin sericin composite membrane; more specifically, the time for the micro-treatment is 15min, 20min, 22min, 26min, 32min, 36min, 47min, 46min, 52min, 54min, 56min or 58 min.
The silk fibroin and sericin composite solution is prepared by adopting a mild degumming and one-step dissolving method, and the traditional method that silk fibroin and sericin are respectively prepared, respectively dissolved and then mixed for preparing a composite material is broken through. Importantly, the molecular weight of silk fibroin in the composite solution is concentrated above 150kDa or contains a molecular weight of about 25kDa, and the molecular weight of the silk fibroin solution prepared by the reported conventional method or the solution prepared by dissolving and mixing silk fibroin sericin is continuously distributed, mainly distributed below 85kDa to 15kDa or even smaller, so that the silk fibroin provided by the invention has the advantages of large molecular weight and small molecular weight dispersion degree, and the strength and the toughness of the composite membrane are improved. Sericin on the outer layer of the silk fibroin fiber is spherical protein, strong hydrogen bond bonding and network bonding between macromolecular chains of the composite membrane are increased due to the existence of the sericin, the strength and toughness of the composite membrane are also improved, and the type and structure of the sericin are different and the performance of the composite membrane is also different along with the outer part and the inner part of a silk fibroin fiber coating layer. According to the invention, the silk fibroin protein composite membrane with different compositions and controllable mechanical properties and high strength and toughness is obtained by controlling the degumming degree, the technical problem of a neck clamp with insufficient mechanical properties of tissue repair materials from natural biopolymers for repairing hard tissues such as dura mater, dura mater and the like is solved, the composite membrane has the activities of inducing cells to rapidly adhere and grow in situ and promoting tissue regeneration, and meanwhile, the composite membrane can be biodegraded and absorbed.
Meanwhile, the application also provides a silk fibroin sericin composite membrane prepared from a silk fibroin sericin composite solution, and the preparation method of the silk fibroin sericin composite solution comprises the following steps:
A) placing silk in warm water for pretreatment, then placing the silk in 65-95 ℃ water for treatment, and drying to obtain primary treated silk;
B) and (3) treating the primarily treated silk in a neutral salt solution, and purifying the obtained silk fibroin and sericin composite dissolved solution to obtain the silk fibroin and sericin composite solution.
The step a) and the step B) are processes for preparing the silk fibroin and sericin composite solution, and are not described herein again. The silk fibroin sericin composite membrane is prepared from a silk fibroin sericin composite solution.
For further understanding of the present invention, the following examples are provided to illustrate the preparation method of the silk fibroin complex solution provided by the present invention, and the scope of the present invention is not limited by the following examples.
Example 1
1. Taking 1 kg of raw silk of silkworms after cocoon cooking and reeling as a raw material, weighing 50L of deionized water according to a bath ratio of 1:50(g/mL) and preheating to 60 ℃, putting the raw silk into the raw silk, stirring the raw silk at a constant temperature of 60 ℃ for 1-10 minutes, taking the raw silk out, washing the silk by using the deionized water at a temperature of 25-30 ℃, dehydrating the silk, and drying the silk by using hot air at a temperature of 40-60 ℃;
2. placing the dried silk in deionized water preheated at 90 ℃ according to a bath ratio of 1:50(g/mL) for boiling for 3 hours at constant temperature, taking out the silk containing sericin, fully cleaning the silk with deionized water at 25-30 ℃, dehydrating, and drying with hot air at 40-60 ℃;
3. the degumming rate of the silk is 14 percent by adopting a sampling and weighing method;
4. weighing dried silkworm silk, dissolving the silkworm silk in 9.3M lithium bromide aqueous solution according to a bath ratio of 1:25(g/mL), and treating at 65 ℃ until the silk is completely dissolved to obtain a silkworm silk fibroin sericin compound solution;
5. filling the silkworm silk fibroin and sericin composite solution into a dialysis bag with the molecular weight cutoff of 14kDa, wherein the wall of the dialysis bag is a semipermeable membrane, placing the dialysis bag filled with the silk fibroin and sericin composite solution into a container filled with deionized water, replacing the water in the container with new deionized water every 2 hours, and continuously dialyzing for 3 days to obtain a purified silkworm silk fibroin and sericin composite aqueous solution;
6. concentrating by adopting a rotary evaporator to obtain a 40mg/mL composite solution, uniformly stirring, removing bubbles in the solution, then pouring into a flat polystyrene plate, carrying out rotary air drying at 25 ℃, taking out, soaking a sample in an 80% ethanol solution for 30-60 min, and immediately placing in a constant temperature and humidity room for air drying to obtain the silk fibroin sericin composite membrane.
7. The tensile property of the silk fibroin sericin composite membrane is measured by using a universal material testing machine: the breaking strength and elongation at break were 12.6MPa and 174%, respectively. The increase in sericin also significantly improves the strength and toughness of the composite film compared with examples 2 and 3.
Example 2
1. Taking 1 kg of raw silk of silkworms after cocoon cooking and reeling as a raw material, weighing 50L of deionized water according to a bath ratio of 1:50(g/mL) and preheating to 60 ℃, putting the raw silk into the raw silk, stirring the raw silk at a constant temperature of 60 ℃ for 1-10 minutes, taking the raw silk out, washing the silk by using the deionized water at a temperature of 25-30 ℃, dehydrating the silk, and drying the silk by using hot air at a temperature of 40-60 ℃;
2. placing the dried silk in deionized water preheated at 90 ℃ according to a bath ratio of 1:50(g/mL) for boiling for 5 hours at constant temperature, taking out the silk containing sericin, fully cleaning the silk with deionized water at 25-30 ℃, dehydrating, and drying with hot air at 40-60 ℃;
3. measuring the degumming rate of the silk by a sampling and weighing method to be 21 percent;
4. weighing dried silkworm silk, dissolving the silkworm silk in 9.3M lithium bromide aqueous solution according to a bath ratio of 1:25(g/mL), and treating at 65 ℃ until the silk is completely dissolved to obtain a silkworm silk fibroin sericin compound solution;
5. filling the silkworm silk fibroin and sericin composite solution into a dialysis bag with the molecular weight cutoff of 14kDa, wherein the wall of the dialysis bag is a semipermeable membrane, placing the dialysis bag filled with the silk fibroin and sericin composite solution into a container filled with deionized water, replacing the water in the container with new deionized water every 2 hours, and continuously dialyzing for 3 days to obtain a purified silkworm silk fibroin and sericin composite aqueous solution;
6. concentrating by adopting a rotary evaporator to obtain a 40mg/mL composite solution, uniformly stirring, removing bubbles in the solution, then pouring into a flat polystyrene plate, carrying out rotary air drying at 25 ℃, taking out, soaking a sample in an 80% ethanol solution for 30-60 min, and then carrying out air drying at a constant temperature and humidity to obtain the silk fibroin sericin composite membrane.
7. The tensile property of the silk fibroin sericin composite membrane is measured by using a universal material testing machine: the breaking strength and elongation at break were 10.5MPa and 127.6%, respectively.
Example 3
1. Taking 1 kg of raw silk of silkworms after cocoon cooking and reeling as a raw material, weighing 50L of deionized water according to a bath ratio of 1:50(g/mL) and preheating to 60 ℃, putting the raw silk into the raw silk, stirring the raw silk at a constant temperature of 60 ℃ for 1-10 minutes, taking the raw silk out, washing the silk by using the deionized water at a temperature of 25-30 ℃, dehydrating the silk, and drying the silk by using hot air at a temperature of 40-60 ℃;
2. placing the dried silk in deionized water preheated at 90 ℃ according to a bath ratio of 1:50(g/mL) for boiling for 7 hours at a constant temperature, taking out the silk containing sericin, fully cleaning the silk with deionized water at 25-30 ℃, dehydrating, and drying with hot air at 40-60 ℃;
3. measuring the degumming rate of the silk by a sampling and weighing method to be 23 percent;
4. weighing dried silkworm silk, dissolving the silkworm silk in 9.3M lithium bromide aqueous solution according to a bath ratio of 1:25(g/mL), and treating at 65 ℃ until the silk is completely dissolved to obtain a silkworm silk fibroin sericin compound solution;
5. the silkworm silk fibroin sericin compound solution is filled in a dialysis bag with the molecular weight cutoff of 14kDa, and the wall of the dialysis bag is a semipermeable membrane. Placing the dialysis bag filled with the silk fibroin sericin composite solution in a container filled with deionized water, replacing the water in the container with new deionized water every 2 hours, and continuously dialyzing for 3 days to obtain a purified silkworm silk fibroin sericin composite aqueous solution;
6. concentrating by rotary evaporator to obtain 40mg/mL composite solution, stirring, removing bubbles in the solution, pouring into flat polystyrene dish, and rotary air drying at 25 deg.C. And taking out, soaking the sample in 80% ethanol solution for 30-60 min, and then air-drying at constant temperature and constant humidity to obtain the silk fibroin sericin composite membrane.
7. The tensile property of the silk fibroin sericin composite membrane is measured by using a universal material testing machine: the breaking strength and elongation at break were 9.6MPa and 113.1%, respectively.
Compared with the examples 2 and 3, the strength and the toughness of the composite membrane are obviously improved by increasing the content of sericin in the silk fibroin and sericin composite solution in the example 1.
Example 4
1. Taking 1 kg of raw silk of silkworms after cocoon cooking and reeling as a raw material, weighing 50L of deionized water according to a bath ratio of 1:50(g/mL) and preheating to 60 ℃, putting the raw silk into the raw silk, stirring the raw silk at a constant temperature of 60 ℃ for 1-10 minutes, taking the raw silk out, washing the silk by using the deionized water at a temperature of 25-30 ℃, dehydrating the silk, and drying the silk by using hot air at a temperature of 40-60 ℃;
2. placing the dried silk in deionized water preheated at 90 ℃ according to a bath ratio of 1:50(g/mL) for boiling for 5 hours at constant temperature, taking out the silk containing sericin, fully cleaning the silk with deionized water at 25-30 ℃, dehydrating, and drying with hot air at 40-60 ℃;
3. measuring the degumming rate of the silk by a sampling and weighing method to be 21 percent;
4. weighing dried silkworm silk, dissolving the silkworm silk in 9.3M lithium bromide aqueous solution according to a bath ratio of 1:25(g/mL), and treating at 65 ℃ until the silk is completely dissolved to obtain a silkworm silk fibroin sericin compound solution;
5. filling the silkworm silk fibroin and sericin composite solution into a dialysis bag with the molecular weight cutoff of 14kDa, wherein the wall of the dialysis bag is a semipermeable membrane, placing the dialysis bag filled with the silk fibroin and sericin composite solution into a container filled with deionized water, replacing the water in the container with new deionized water every 2 hours, and continuously dialyzing for 3 days to obtain a purified silkworm silk fibroin and sericin composite aqueous solution;
6. concentrating by rotary evaporator to obtain 60mg/mL composite solution, stirring, removing bubbles in the solution, pouring into flat polystyrene dish, and rotary air drying at 25 deg.C. And taking out, soaking the sample in 80% ethanol solution for 30-60 min, and then air-drying at constant temperature and constant humidity to obtain the silk fibroin sericin composite membrane.
7. The tensile property of the silk fibroin sericin composite membrane is measured by using a universal material testing machine: the breaking strength and elongation at break were 12.9MPa and 162.4%, respectively.
Comparative example 1 preparation method of conventional fibroin solution
1. The method comprises the following steps of taking raw silkworm silks which are reeled by boiling cocoons as raw materials, putting the raw silkworm silks into a sodium carbonate aqueous solution with the concentration of 0.1-0.06% according to the bath ratio of 1:50(g/mL), treating for three times at the temperature of 98-100 ℃ for 30 minutes each time, then fully cleaning the silks with deionized water at the temperature of 25-30 ℃, dehydrating, and drying with hot air at the temperature of 40-60 ℃;
2. measuring the degumming rate of the silk by a sampling and weighing method to be 23 percent;
3. weighing dried silkworm silk, dissolving the dried silkworm silk in 9.3M lithium bromide aqueous solution according to a bath ratio of 1:25(g/mL), and treating at 65 ℃ until the silk is completely dissolved to obtain silkworm fibroin solution;
4. filling silkworm silk fibroin solution into a dialysis bag with the molecular weight cutoff of 14kDa, wherein the wall of the dialysis bag is a semipermeable membrane, placing the dialysis bag filled with the silk fibroin solution into a container filled with deionized water, replacing the water in the container with new deionized water every 2 hours, and continuously dialyzing for 3 days to obtain a purified silkworm silk fibroin aqueous solution;
5. concentrating by adopting a rotary evaporator to obtain 40mg/mL silk fibroin aqueous solution, uniformly stirring, removing bubbles in the solution, then pouring into a flat polystyrene plate, carrying out rotary air drying at 25 ℃, taking out, soaking a sample by using 80% ethanol solution for 30-60 min, and then carrying out air drying at constant temperature and constant humidity to obtain the silk fibroin membrane.
6. The tensile property of the silk fibroin film is measured by using a universal material testing machine: the breaking strength is 6.0MPa, and the breaking elongation is 58%.
Comparative example 2 composite membrane prepared by mixing conventional fibroin solution preparation method with sericin
1. The method comprises the following steps of taking raw silkworm silks which are reeled by boiling cocoons as raw materials, putting the raw silkworm silks into a sodium carbonate aqueous solution with the concentration of 0.1-0.06% according to the bath ratio of 1:50(g/mL), treating for three times at the temperature of 98-100 ℃ for 30 minutes each time, then fully cleaning the silks with deionized water at the temperature of 25-30 ℃, dehydrating, and drying with hot air at the temperature of 40-60 ℃;
2. measuring the degumming rate of the silk by a sampling and weighing method to be 23 percent;
3. weighing dried silkworm silk, dissolving the dried silkworm silk in 9.3M lithium bromide aqueous solution according to a bath ratio of 1:25(g/mL), and treating at 65 ℃ until the silk is completely dissolved to obtain silkworm fibroin solution;
4. filling silkworm silk fibroin solution into a dialysis bag with the molecular weight cutoff of 14kDa, wherein the wall of the dialysis bag is a semipermeable membrane, placing the dialysis bag filled with the silk fibroin solution into a container filled with deionized water, replacing the water in the container with new deionized water every 2 hours, and continuously dialyzing for 3 days to obtain a purified silkworm silk fibroin aqueous solution;
5. taking 1 kg of raw silk of the silkworm reeled by boiling the cocoon, weighing 50L of deionized water according to a bath ratio of 1:50(g/mL) and preheating to 60 ℃, putting the raw silk into the deionized water and stirring at the constant temperature of 60 ℃ for 1-10 minutes, taking out the raw silk and washing and dehydrating the silk by using deionized water at the temperature of less than 25-30 ℃;
6. placing the dehydrated silk in preheated deionized water at 90 ℃ according to a bath ratio of 1:50(g/mL) for boiling for 7 hours at a constant temperature, removing fibers, collecting a sericin water solution, and freeze-drying to obtain solid sericin;
7. and (3) concentrating the silk fibroin aqueous solution obtained in the step (4) by adopting a rotary evaporator, adding sericin with the same sericin content as that in the example (4) and prepared in the step (6), dissolving and mixing uniformly, adjusting the concentration of the silk fibroin sericin composite solution to be 60mg/mL, removing bubbles in the solution, then pouring the solution into a flat polystyrene plate, carrying out rotary air drying at 25 ℃, taking out the solution, soaking the sample for 30-60 min by using 80% ethanol solution, and then carrying out air drying at constant temperature and constant humidity to obtain the silk fibroin sericin composite membrane.
8. The tensile properties of the silk fibroin films were measured using a universal material tester as follows: the breaking strength is 6.83MPa, and the breaking elongation is 65%.
The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (9)
1. A preparation method of a silk fibroin sericin composite membrane comprises the following steps:
A) placing silk in warm water for pretreatment, then placing the silk in 65-95 ℃ water for treatment, and drying to obtain primary treated silk; the temperature of the warm water is 35-60 ℃;
B) treating the primarily treated silk in a neutral salt solution, and purifying the obtained silk fibroin sericin composite dissolved solution to obtain a silk fibroin sericin composite solution;
C) concentrating the silk fibroin and sericin composite solution, forming a film, placing the obtained initial composite film in micromolecular monohydric alcohol for micro-treatment, and then cleaning and drying to obtain a silk fibroin and sericin composite film;
the neutral salt solution is a lithium bromide aqueous solution with the concentration of 8-10M, and the ratio of the primarily treated silk to the lithium bromide aqueous solution is 1 g: (20-30) mL.
2. The method according to claim 1, wherein the pretreatment time is 1 to 10 min.
3. The preparation method according to claim 1, wherein in the step A), the treatment time in the water at 65-95 ℃ is 1-8 h.
4. The method according to claim 1, wherein in step a), the ratio of silk to warm water is 1 g: (40-60) mL, wherein the ratio of the pretreated silk to water is 1 g: (40-60) mL.
5. The method according to claim 1, wherein the step a) further comprises, after the pretreatment: taking out the pretreated silk, washing in water at 25-30 ℃, and dehydrating;
the method also comprises the following steps of: washing the treated silk in water at 25-30 ℃ and then dehydrating; the drying is carried out by hot air at 40-60 ℃.
6. The method according to claim 1, wherein the temperature of the treatment in step B) is 50 to 80 ℃.
7. The preparation method according to claim 1, wherein the purification method is specifically:
and (3) filling the obtained silk fibroin sericin composite dissolved solution into a dialysis bag with the molecular weight cutoff of 10-50 kDa, and placing the dialysis bag in deionized water for continuous dialysis for 1-3 days.
8. The method according to claim 1, wherein in step C), the small molecule monohydric alcohol is an ethanol solution with a concentration of 70-80%, and the micro-treatment time is 10-60 min.
9. A silk fibroin sericin composite membrane is prepared from a silk fibroin sericin composite solution, and the preparation method of the silk fibroin sericin composite solution comprises the following steps:
A) placing silk in warm water for pretreatment, then placing the silk in 65-95 ℃ water for treatment, and drying to obtain primary treated silk; the temperature of the warm water is 35-60 ℃;
B) treating the primarily treated silk in a neutral salt solution, and purifying the obtained silk fibroin sericin composite dissolved solution to obtain a silk fibroin sericin composite solution;
the neutral salt solution is a lithium bromide aqueous solution with the concentration of 8-10M, and the ratio of the primarily treated silk to the lithium bromide aqueous solution is 1 g: (20-30) mL.
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